| Objective:1)This research was on the polymorphism detection of Major Histrocompitbility complex Classâ… releted gene A (MICA) and determine the mechanism of MICA antibodies in liver transplantation rejection. The total experiment includes following sections: 1) Inspect the serum of patients who underwent liver transplantation with MICA tansfected cell line, detect patients serum with flow cytomertry for finding whether it had MICA antibodies or not, then determine the mechanism of MICA antibodies in liver transplantation rejection.2)Using PCR method to detect the MICA alleles in serum of 22 cases in both donors and recipients who underwent liver transplantation rejection, and identify the mechanism of the MICA*008 allele in liver transplantation rejection.3)Using the method of IHC to further verifying the methanism between MICA antigens and liver transplantation rejection.Method:First part of experement:1)Test the transfected human B lymphoblastic cell line (721. 221) after cell culturing with serum of 84 cases who underwent liver transplantation, all the experiment were analysized with flow cytomertry. The experiment was identified as following groups, Group one: MICA transfected cells (the amount of cells are half million per sample) +goat anti mouse antibody, 2 nl (second antibody, also fluorescent antibody); Group two: MICA transfected cells (the amount of cells are half million per sample) +anti-MICA antibody(first antibody, also fluorescent antibody)+ goat anti mouse antibody, 2 nl(second antibody, also fluorescent antibody); Group three: MICA transfected cells ( the amount of cells are half million per sample) + the AB type sera for none- history of blood transfusion patients were considered as the negative control+goat anti human (IgG+IgM) 10nl (second antibody); Group four: MICA transfected cells (the amount of cells are half million per sample) + the serum of the patients who accepted blood transfusion several time or underwent organ transplantation and produced alloantibody, were classified as the positive control+goat anti human (IgG+IgM) 10nl (second antibody); Group five: MICA transfected cells (the amount of cells are half million per sample) + anti-MICA antibody (first antibody, also fluorescent antibody) +the serum of the patients who had the record of liver transplantation rejection+goat anti human (IgG+IgM) 10nl (second antibody); MICA monoclonal antibody was used to block in case of finding whether there are any MICA special antibody in serum of patients in all groups.2) MICA transfected cells?BEC, LSEC and hepatocyte were conjugated with 5nl anti-MICA antibody, added goat anti mouse antibody (fluorescent antibody), and check them with flow cytomertry respectively. MICA none-transfected cells and transfected cells were considered as negative and positive control respectively.Second part of experiment:1) The DNA of serum for 22 cases of donors and recepients who had record of liver transplantation rejection were tested with spectrophotometer respectively for the case of finding whether there were enoght amount of DNA meet the requirement for PCR detection. Then tested them with MICA special primer through PCR method, the electrophoretogram was analysed and record, and every MICA allels genotype were obtained from contrast with MICA allels genotype map as control.2) BEC, LSEC and hepatocyte were taken out from human health liver respectively, cells were cultured for a few days, RNA was taken out from them and synthesis for cDNA. G6PD was considered as endogenetic normal control, MICA first generation cell line and MICA transfected cells were regarded as negative and positive control respectively, tested them with RT-PCR and Western-blot then.Third part of experiment:13 cases who had record of liver transplantation rejection and 15 cases who underwent liver transplantation but no episode of rejection were taken liver biopsy at certain time point respectively. Those who had no record of rejection were applied as positive control. Again those who suffered colon cancer were taken colon section and applied as negative control. All samples were tested with IHC method.Results:1) A total of 22/84 (26%) patients with MICA antibodies were observed either pre and/or post-transplantation. 8/84 (9.5%) patients had MICA antibodies in the pre- and 14/84 (17%) in the post-transplantation period. Overall 43/84 (51%) patients had rejections. No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibodies as compared to those without MICA antibodies (29/62, 47% p=ns). There were no MICA expression in none-MICA transfected cells, however, there were highly MICA expression in MICA tranfected cells. After blocked with MICA monoclonal antibody, the conjugation between serum and MICA tranfected cells was inhibitted, suggests these detected antibodies in rerum of patients were MICA special antibodies. In MICA antibody negative and positive groups, regardless of whether there were rejection or not and patients'survival or not, there were not any clinical relation between two groups.2)From the results of the folw cytomertry, there were MICA expression on the surface of MICA transfected cells, however, as the negative control, there were no any expression on the surface of BEC, LSEC and hepatocyte, suggests that the MICA were not expressed on the surface of these cells.3) The most frequenct MICA allels occurring in both donors and recipients were MICA*008. In 9 patient's serum both donors and recipients the MICA*008 allel could be detected, 4 of them were completely the same in MICA*008 typing; In 4 cases, donors were not cross-match, but their alleles are MICA*008. In 5 cases, the MICA alleles were 002 or 010 in donors without cross-match typing. In 14/22 (63%) patients who have suffered rejection, MICA*008 could be detected, but in 29/62 (47%) patients, MICA*008 could not be detected, there is no significant differentiation between them (P>0.05).4) Testing the fresh liver cells and anti-MICA monoclonal antibody with Western-blot, shown that 62kDa band which observed was corresponging with MICA multipeptide. These results were solely detected in MICA transfected cell, not in liver cells, such as BEC, LSEC and hepatocyte.5) MICA antigens can be slightly expressed in liver biopsy sections of who underwent liver transplantation and the patients who had rejection records at cerntain time point. However, there were quite strong and diffusing MICA expression was observed in section of those who suffered colon cancer.Conclusion:1) MICA antibody can be solely expressed on the surface of MICA tranfected cell and no expression can be found on the surface of other liver cells, such as BEC, LSEC and hepatocyte.2) MICA antigen can be slightly expressed in the hepatic tissues of those who underwent liver transplantation and/or had records of rejection, however, strongly expressed in the tissues of those who suffered colon cancer.3) From the statistical results, in pre-existed antibodies in drafted liver, no MICA allels were observed. Therefore, MICA may not be the reasoning and target MHC gene for liver transplantation rejection compared with kidney transplantation.
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